Inflammation is the reaction of an organism to injury and an initiator of the healing process. Local symptoms of inflammation include heat (calor), redness (rubor), swelling (tumor) and pain (dolor), and may be readily apparent on physical examination of a patient. Acute inflammation, the primary mechanism of host immune protection against infectious or harmful agents, is the consequence of complex ligand-receptor interactions involving the humoral and phagocyte components of immunity. Systemic inflammation is not as easy to identify and/or quantify as local inflammation. Fever and leukocytosis (i.e., transient increase in the number of leukocytes in the blood) are the most prominent systemic manifestations of acute inflammation. The leukocytosis is a neutrophilia (i.e., increase in the number of polymorphonuclear neutrophil leukocytes or PMNLs) in most bacterial infections, and an eosinophilia (i.e., increase in the number of eosinophils) in most allergic diseases and parasitic infestations. Many viral diseases are associated with relative or absolute lymphocytosis (i.e., increase in the number of lymphocytes), whereas certain viral, richettsial, bacterial, and protozoan infections are characterized by leukopenia (i.e., a reduction in the number of leukocytes). The intensity of the inflammatory response is related to the severity of injury and the humoral/immune system reaction capacity of the host.
The immunoglobulin dependent and independent activation of the classical and alternative pathways, respectively, of complement provide the principle humoral effector mechanisms of inflammation. Activation of either pathway results in a cascade of proteolytic activities generating anaphylatoxins, i.e., freely diffusible ligands such as the relatively low molecular weight complement fragments, C5a and C3a. This activity also generates opsonin. The larger molecular weight complement fragment C3b is an opsonin that covalently reacts with and fixes to a broad variety of molecules present on microbial surfaces, and prepares the surfaces for attack by phagocytes (a process referred to as opsonification).
Although endothelial cells, platelets, eosinophils, basophils and lymphocytes play specialized roles, the polymorphonuclear neutrophil leukocyte (PMNL) and monocyte are the principle cellular effectors of the acute inflammatory response. The humoral-phagocyte relationship can be conceptualized as an information-effector mechanism for rapid response to infection or injury. The complement system responds to general classes of foreign substances, e.g., certain polysaccharides, as well as specific antigens via antigen-specific immunoglobulins. The location and magnitude of the infection or injury is transmitted to the various cellular effectors of inflammation via an enzymatically amplified cascade of protolytic activity. The smaller molecular weight anaphylatoxins produced (e.g., C5a, as well as certain bacterial peptide markers of bacterial infection, such as N-formyl methionyl peptides (e.g., N-fMLP), diffuse from the site of generation inducing the localized tissue changes characteristic of inflammation and serving to establish a chemotactic concentration gradient. The opsonic products of activation (e.g., C3b) bind to the initiator substance, e.g., a microbe. As a result, local hemodynamic changes and endothelial alterations promote phagocyte margination, sticking, diapedesis, and migration to the site of inflammation where contact with the complement and/or immunoglobulin opsonified foreign substance, results in 1) phagocyte recognition of the opsonified material as a foreign substance via opsonin-specific receptors, 2) engulfment i.e., phagocytosis, and 3) the activation of microbicidal metabolism.
Microbicidal metabolism is characterized by nonmitochondrial O.sub.2 consumption and glucose dehydrogenation via the hexose monophosphate shunt (Sbarra and Karnovsky, 1960, J. Biol. Chem. 234:1355). These activities reflect the activation of NAD(P)H oxidase (Rossi et al., J. Reticuloendothel. Soc. 12:127) yielding reduced oxygen products that can participate in microbicidal reactions (Review: Badwey and Karnovsky, 1980, Annu. Rev. Biochem. 49:695). Chemiluminescence, i.e., photon emission, is an energy product of these oxygenation reactions (Allen, 1972, Biochem. Biophys. Res. Commun. 47:679). The chemiluminescence quantum yield, i.e., the ratio of photons emitted per oxygenation event, is dependent on the type and quantity of generated oxygenating agents and the nature of the substrate oxygenated. The native luminescence product of phagocyte microbicidal action reflects the oxygenation of natural substrates presented. Such reactions are of relatively low quantum yield and vary with the nature of the substrate oxygenated. Introduction of exogenous high quantum yield chemiluminigenic substrates (CLS) overcomes the problems of variability and sensitivity. Luminol and other cyclic hydrazides increase quantum yield by greater than three orders of magnitude and at the same time impose control with regard to the substrate oxygenation measured (Allen and Loose, 1976, Biochem. Biophys. Res. Commun. 69:245). Acridinium compounds such as lucigenin can also be employed as CLS (Allen, 1981, in Bioluminescence and Chemiluminescence: Basic Chemistry and Analytical Applications, DeLuca and McElroy, eds., pp. 63-73, Academic Press, New York). However, these CLSs differ with regard to the type of oxygenation activity measured (Allen, 1982, in Chemical and Biological Generation of Excited States, Adam and Cilento, eds., pp. 309-344, Academic Press, New York, and Allen, 1986in Meth. Enzymol. 133:449).
Complement activation plays the principle role in precipitating the acute inflammatory response. The complement system is composed of 20 different proteins that account for approximately 15% of the plasma globulins. In the past, measurement of the various complement proteins, products of activation, and functional capacity have provided the major avenues of approach to estimating the state of systemic inflammation (See: Cooper, 1986, in Clinics in Laboratory Medicine: Advances in Immunopathology, Volume 6, Number 1, Nakamura and Rowlands, eds., pp. 139-155, W. B. Saunders Comp., Philadelphia). Until recently, virtually all complement system testing was based on: 1) measuring the collective functionality of the system, e.g., CH.sub.5O hemolytic assay, or 2) quantification of complement components, e.g., C3, C4, factor B. These techniques are limited in that they only measure the static plasma levels of specific complement system components, and not the dynamics of complement activation. Since synthesis is quite responsive to consumption and several components behave as acute phase reactants, these measurements are less than ideal for evaluating the state of inflammation of a patient.
More recently, several methodologies have been directed to detecting and quantifying the complement activation process. These activation-specific complement assays detect physical, chemical, or antigenic changes in complement components consequent to in vivo activation. The three general assay approaches used are based on detecting: 1) proteolytic alteration of a complement component, 2) alteration in antigenicity, or 3) protein-protein complexes that form as a result of complement activation.
Assays based on detecting proteolytic alteration of a complement component include the determination of C4d/C4 and C3d/C3 ratios. In a typical assay of this type, proteolytic product, e.g., C4d, is isolated from the residual parent component, e.g., C4, by electrophoresis, and both parent and product molecules are determined immunochemically, e.g., by rocket immunoelectrophoresis (See Curd, 1982, in Analysis and Recent Progress in Diagnostic Laboratory Immunology, Nakamura et al., eds., pp. 215-230, Masson Publ., Nurnberger and Bhadki, 1984, J. Immunol. Meth. 74:87). This approach is technically involved, time consuming, and relatively insensitive.
Radioimmunoassay of the anaphylatoxins C3a, C4a, and C5a provides a more sensitive methodology belonging to this first category (See Gorski, 1981, J. Immunol. Meth. 47:61; Hugli and Chenoweth, 1980, in Future Perspectives in Clinical Laboratory Immunoassays, Nakamura et al., eds., pp. 443-460, Alan R. Liss, Inc., New York). However, this approach is also technically involved, time consuming, and requires the use of radioisotopes.
An example in the second category of activation-specific assays (i.e., where an alteration in antigenicity is determined) is the disappearance of C1r but retention of C1s antigen expression following complement activation. This differential loss results from C1-inhibitor (C1In) binding. As such, the ratio of simultaneously determined C1r:C1s provides an expression of C1 activation (Ziccardi and Cooper, 1978, J. Exp. Med. 147:385) as well as C1In functional capacity of plasma (Ziccardi and Cooper, 1980, Clin. Immunol. Immunopathol. 15:465).
The third category of activation-specific assays relates to the determination of protein-protein complexes that form as a result of complement activation. Protein-protein complexes are characteristic of both classical, e.g., C1r-C1s-C1In, and alternative, e.g., Bb-C3bn-P, activation pathways. For example, in the Bb-C3bn-P complex enzyme-linked immunosorbent assay (ELISA) for detection of alternative pathway activation, a sample is contacted with surface-bound antiprotein P antibody which binds the complex as well as free protein P. After washing, the bound material is further contacted with anti-C3 antibody enzyme conjugate indicator system. Following washing and incubation with a substrate for color development, the degree of color development is related to the quantity of C3bn-P complex in the sample (Cooper et al., 1983, Springer Sem. Immunopathol. 6:195).
Although, the foregoing activation-specific complement system assays provide additional information regarding the status of specific complement system components in a sample, their complexity, lack of sensitivity and, in some cases, requirement of radioactive materials has prevented their widespread adoption and use for routine clinical diagnostic purposes. Thus, a need exists for improved methods for evaluating the in vivo status of the inflammatory response of a patient which overcomes the problems associated with prior art processes and reagents.